PROJECT SUMMARY The overall objective of the studies proposed in this revised R01 application is to provide direct evidence that OT neurons are organized into parallel pathways, which have discrete projection targets that subserve domain specific social behaviors; a first step in a larger project aimed at identifying circuit based approaches which could act as therapeutics for treating ASD. Based on extensive preliminary findings, our overall hypothesis is that functional specializations of magnocellular and parvocellular subtypes of oxytocin neurons dictate the specificity of distinct behaviors thought to underlie domain specific social impairments in ASD. Moreover, preliminary data suggest that these pathogenic mechanisms will be differentially impacted by therapeutic interventions such as intranasal oxytocin (currently in clinical trials for ASD), and highlight the importance of these studies for future trials that seek to stratify patients based on a more complete understanding of the pathogenesis of social deficits in autism. Despite our important preliminary findings, our hypothesis is based solely on studies that have examined the contribution of OT neurons and circuits to social functions which have treated these neurons as an undifferentiated group, in part because of technical limitations in specifying mOT and pOT neurons for functional genomics. For example, we have previously demonstrated that bath application of OT evokes a novel form of synaptic plasticity in the NAc (OT-LTD), and correlated this cellular mechanism to social (peer) reward learning, a behavior that is blocked by antagonists or molecular ablation of OTRs in the NAc (Dlen et al., 2013). Nevertheless pharmacologically induced OT-LTD, as well as knockdown or pharmacological blockade of OT receptors (OTRs), cannot distinguish between magnocellular and parvocellular mechanisms. The goal of this R01 application is to explore this understudied area and to develop tools to test our hypothesis directly in vivo and ex vivo, and provide the basis and means for further studies on the specific role of oxytocin in circuit based defects observed in ASD. Three specific aims have been proposed to achieve our goal. Specifically: Specific Aim 1: To delineate the electrophysiological properties of NAc projecting OT neurons in the hypothalamus. Here we will test the hypothesis that the NAc receives an exclusively parvocellular OT projection. Specific Aim 2: To determine whether OTergic inputs are able to evoke synaptic plasticity in the NAc. Here we will test the hypothesis that OT induced synaptic plasticity in the NAc is mediated by OT released from axonal projections from the hypothalamus. Specific Aim 3: To determine the functional domain specificity of the OT projection to the NAc. Here we will test the hypothesis that the OTergic projection to the NAc is required for social (peer), but not filial (pup) or drug (cocaine) reward learning. In addition, the studies proposed in Aims 1-3 will deliver several novel tools for functional genomics, including: the OT-Flp KI mouse, CAV-FonCre virus, the AAV-FonChronos virus, and the pup CPP assay to measure the rewarding properties of filial attachments. These studies will add to the growing evidence that OTergic circuits are organized into parallel processing circuits, as is seen elsewhere in the brain. In addition, the current studies will lay the groundwork for future development of multiple feature selection tools that will allow us to interrogate the contribution of mOT functions, in brain regions where we have identified mixed mOT and pOT inputs (e.g. VTA, (Hung et al., 2017)). Taken together, these studies will uncover basic mechanisms underlying ASD relevant social circuits, using a quantifiable measure of social reward learning, which can be used in both humans and mice, dramatically improving the translational validity of these studies for future development of therapeutic strategies in ASD.